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Online Laboratory Investigations

Trigeminal Nerve Stimulation: A Novel Method of Resuscitation for Hemorrhagic Shock*

Li, Chunyan PhD1,2; Chiluwal, Amrit MD1; Afridi, Adil MS2; Chaung, Wayne PhD3; Powell, Keren BA2; Yang, Weng-Lang PhD3; Wang, Ping MD3; Narayan, Raj K. MD1,2

Author Information
doi: 10.1097/CCM.0000000000003735

Abstract

Hemorrhagic shock (HS) is the leading cause of preventable deaths among trauma victims, accounting for around 40% of mortality; 33–56% of these deaths happen in the prehospital period (1–3). Both complete cessation of bleeding and restoration of the intravascular volume via blood product transfusion are essential in treating patients experiencing HS (45). However, there are situations when bleeding cannot be completely stopped and massive blood transfusion cannot be initiated promptly, such as when patients suffer serious traumatic injuries in locations remote from medical facilities (e.g., traffic accidents or battlefields). In such situations, any ability to exploit the body’s natural compensatory mechanism to prevent total circulatory collapse until the patient can be transported to a trauma center would be extremely invaluable (6).

The compensatory response to HS is driven by the autonomic nervous system (ANS) (7). It depends on a complex interplay between the sympathetic nervous system (SNS) and parasympathetic nervous systems, with the SNS playing a dominant role. Previous studies in animal models involving direct measurement of activity in nerves supplying renal, adrenal, splenic, hepatic, and cardiac tissues (8–12), as well as indirect (heart rate variability [HRV] and microneurography) (1314) recording of neural activity have shown that the SNS plays a crucial role in hemodynamic compensation after HS. Activation of the SNS after severe HS results in cardiac stimulation, reduction of the venous capacity, selective peripheral vasoconstriction, and increased transcapillary fluid influx in its attempt to maintain blood pressure (BP) and preserve blood flow to vital organs.

Neuromodulation of the SNS has never been studied as a target for resuscitation after HS. This may be due in part to the difficulty in modulating the sympathetic fibers in a trauma field care scenario. However, we have previously shown that percutaneous trigeminal nerve stimulation (TNS) modulates the SNS (15). Thus, in this study, we hypothesize that TNS treatment acts on the SNS pathway and rebalances the ANS, leading to longer survival time following HS. To test our hypothesis, we established a severe model of HS using rats and explored the role of TNS on survival rate, ANS activity, hemodynamics, brain perfusion, catecholamine release, and systemic inflammation. Although there has been a tremendous interest in fluid resuscitation and vasoactive agents in the management of HS, modulation of the innate sympathetic and parasympathetic compensatory mechanism via TNS has not been studied thus far. Such an approach, if successful, could represent a paradigm shift in the way we treat severe HS, especially in the critical early minutes and hours after the injury.

METHODS

Animal Experiments

Male Sprague-Dawley rats (275–325 g) underwent severe HS as detailed in Supplemental Digital Content 1 (http://links.lww.com/CCM/E429) and Supplemental Digital Content 2 (http://links.lww.com/CCM/E430; legend, Supplemental Digital Content 1, http://links.lww.com/CCM/E429). Animals were randomly assigned to four groups: HS control (control no. 1: neurectomy of trigeminal nerve branches; control no. 2: nonspecific stimulation of facial nerves) (Supplemental Digital Content 3, http://links.lww.com/CCM/E431; legend, Supplemental Digital Content 1, http://links.lww.com/CCM/E429), HS vehicle (vehicle), and HS with TNS treatment groups (TNS). All groups underwent the same surgery procedures, including placing electrodes for TNS; however, stimulation was not applied to the vehicle group. Methods of TNS, brain probe implantation (1617), HRV analysis (1819), and measurement of blood norepinephrine and cytokines are detailed in Supplemental Digital Content 1 (http://links.lww.com/CCM/E429). All experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research.

Statistics

All data are expressed as mean ± sd and analyzed by SigmaStat software. Survival rate analysis was performed by the Kaplan-Meier method and compared by the log-rank test. The mean arterial BP (MAP), heart rate (HR), HRV, cerebral blood flow (CBF), and brain oxygen tension (Pbro2) were analyzed using one-way repeated measures analysis of variance (ANOVA) for the same group at different time points. The difference between multiple groups was analyzed by two-way repeated measures ANOVA and all pairwise multiple comparison procedures (Student-Newman-Keuls method) by setting group (vehicle vs TNS) and time (baseline, 0, 15, 30, and 45 min) as the two factors. Student t test was used when only two groups were compared. p values of less than 0.05 were considered significant.

RESULTS

TNS Improved Survival Without Fluid Resuscitation

Following severe HS, rat survival was monitored for 2 hours. The percentages of blood volume withdrawn per body weight necessary to induce HS were similar between control no. 1, control no. 2, vehicle, and TNS treatment groups, 50.1% ± 1.6%, 50.0% ± 1.2%, 50.1% ± 1.9%, and 51.3% ± 1.8%, respectively (Fig. 1A). We tested whether treatment with TNS can prolong survival following severe HS without fluid resuscitation. In the control no. 1, control no. 2, and TNS treatment groups, intermittent TNS was given for ` hour immediately following the shock period. MAP did not increase in both control groups, whereas it consistently increased in the TNS treatment group (Supplemental Digital Content4, http://links.lww.com/CCM/E432; legend, Supplemental Digital Content 1, http://links.lww.com/CCM/E429). Furthermore, TNS triggered sympathetic nerve activity driven low-frequency (LF) oscillations of BP, which have been shown to be highly associated with an increased tolerance to central hypovolemia (Supplemental Digital Content 5, http://links.lww.com/CCM/E433; legend, Supplemental Digital Content 1, http://links.lww.com/CCM/E429). In the vehicle group, the mean survival time after severe HS was 50 minutes, with 95% CIs ranging from 43.8 to 56.2 minutes. Similar mean survival time was observed for control no. 1 and control no. 2 groups, 50.6 minutes and 51.4 minutes, respectively. In the TNS treatment group, however, the mean survival time was 101.3 minutes, with 95% CI ranging from 92.4 minutes to 110.1 minutes (Fig. 1B). None of the rats survived more than 60 minutes (n = 15) in the vehicle, control no. 1, and control no. 2 groups. With TNS treatment, the survival rate was 90% at 60 minutes and 35% at 120 minutes (n = 20; p < 0.001) (Fig. 1B). This result shows that TNS treatment significantly increased the short-term survival of rats following severe HS.

Figure 1.
Figure 1.:
Effects of trigeminal nerve stimulation (TNS) on survival rate without fluid resuscitation. A, The total amount of blood withdrawn (volume of blood withdrawn/body weight, %) from the hemorrhagic shock (HS) vehicle, TNS treatment, Control (Cont) # 1, and Cont # 2 groups are shown in the box plot. The box plot shows the median, 10th, 25th, 75th, and 90th percentile as vertical box with error bar. There is no significant difference between all four groups. B, Alteration in the survival rates at 120 min were analyzed. The survival rates were estimated by the Kaplan-Meier method and compared by using the log-rank test. * p < 0.001 versus vehicle, n = 15 for vehicle group, n = 20 for TNS treatment group, n = 8 for Cont # 1 group (neurectomy of trigeminal nerve branches), and n = 7 for Cont # 2 group (nonspecific stimulation of facial nerves).

Effect of TNS on ANS Following Severe HS

To determine the effect of TNS on ANS activity, HRV was analyzed from the electrocardiogram recordings (details described in Supplemental Digital Content 1, http://links.lww.com/CCM/E429). The ANS responses to the first cycle of TNS immediately after hemorrhage are shown in Supplemental Digital Content 6 (http://links.lww.com/CCM/E434; legend, Supplemental Digital Content 1, http://links.lww.com/CCM/E429). LF/total is an index of SNS tone, whereas high frequency (HF)/total is an index of PNS tone (1819). At TNS-on phase, LF/total was 2.7× higher than the baseline (100%), whereas HF/total was decreased by 50% (n = 6). However, at TNS-off phase, LF/total had dropped by 60%, HF/total had increased 1.3× compared with the baseline, and MAP was maintained at a high level. In the vehicle group, there were no noticeable changes in LF/total and HF/total compared with the baseline, when plotted on the same scale as TNS treatment. At TNS-off phase, the average LF/total of TNS treatment was 57% lower, whereas the average HF/total of TNS treatment was 17% higher when compared with the vehicle group. Induction of HS resulted in significant changes in ANS activity (Fig. 2, A and B). There were no significant differences in baseline measurements and during HS period between vehicle and TNS groups. LF/total increased dramatically during shock period, whereas HF/total decreased. With TNS treatment, LF/total was significantly lower when compared with the vehicle group during the observation period. Furthermore, HF/total was higher in the TNS treatment group than the vehicle group. At 30 and 45 minutes after HS, LF/total decreased 2.60× and 3.66×, whereas HF/total increased 1.22× and 1.19× compared with the vehicle group. These data demonstrate that TNS modulates ANS activity and can attenuate HS-induced SNS hyperactivity without volume expansion with fluid resuscitation.

Figure 2.
Figure 2.:
Effects of trigeminal nerve stimulation (TNS) on autonomic nervous system activities. A, During shock period, low-frequency (LF)/total increased dramatically. With TNS treatment, the LF/total ratio was significantly lower when compared with the vehicle group during the observation period. B, High-frequency (HF)/total decreased dramatically during shock period. With TNS treatment, HF/total was significantly higher in the TNS treatment group than the vehicle group at 30 and 45 min. Data were expressed as mean ± sd and analyzed using one-way and two-way repeated measures analysis of variance. # p < 0.001 versus HS onset (0 min) in TNS group, % p < 0.001 versus HS onset (0 min) in vehicle group, * p < 0.05 versus vehicle at the same time point, n = 6~8/group.

Effect of TNS on Hemodynamics Following Severe HS

The hemodynamic variables of rats which survived longer than 45 minutes for the vehicle group and 120 minutes for the TNS group were compared. We compared MAP and HR between two groups at 15, 30, and 45 minutes after HS. The baseline MAP (92.3 ± 2.5 mm Hg vehicle vs 94.7 ± 4.8 mm Hg TNS) was similar between two groups. At 15, 30, and 45 minutes after HS, MAP in the TNS treatment group was significantly higher than the vehicle group, 35.3%, 51.8%, and 60.2%, respectively (Fig. 3A). However, there was no significant difference in HR between the vehicle and TNS treatment groups (Fig. 3B). These results suggest that TNS improves hemodynamics during the critical early minutes and hours following severe HS. These enhanced hemodynamic variables are important factors for survival, as well as in attenuating end organ injury.

Figure 3.
Figure 3.:
Effects of trigeminal nerve stimulation (TNS) on hemodynamics. A, Mean arterial blood pressure (MAP). B, Heart rate. TNS treatment significantly increased MAP in comparison with the vehicle group at 15, 30, and 45 min after severe hemorrhagic shock (HS). There were no significant changes in heart rates at all the time points. Data were expressed as mean ± sd and analyzed using one-way and two-way repeated measures analysis of variance. # p < 0.001 versus HS onset (0 min) in TNS group, % p < 0.001 versus HS onset (0 min) in the vehicle group, * p < 0.05 versus vehicle at the same time point, n = 6~8/group. BPM = beats/min.

Effect of TNS on Cerebral Perfusion Following Severe HS

Understanding cerebral hemodynamic responses to blood loss is an essential target for improving survival to HS. Representative recordings and changes in cerebral variables after severe HS are shown in Supplemental Digital Content 7 (http://links.lww.com/CCM/E435; legend, Supplemental Digital Content 1, http://links.lww.com/CCM/E429) and Fig. 4. In both vehicle and TNS groups, CBF decreased significantly by 45.1% and 45.4% at HS onset (0 min), and Pbro2 decreased by 35.9% and 37.3%, respectively, when compared with the baseline (–40 min). During the observation period, for the vehicle group, CBF and Pbro2 changed from 27.9 ± 1.9 to 35.5 ± 3.4 mL/min/100 g and from 12.3 ± 2.0 to 12.9 ± 2.2 mm Hg, respectively, at 30 minutes after HS in comparison with HS onset. With TNS treatment, CBF and Pbro2 increased by 37.9% (48.3 ± 3.2 vs 35.5 ± 3.4 mL/min/100 g) and 41.4% (18.6 ± 1.5 vs 12.9 ± 2.2 mm Hg) in comparison with vehicle group at 30 minutes after HS. These observations suggest that TNS treatment has a neuroprotective effect against hypoxic-ischemic injury after severe HS.

Figure 4.
Figure 4.:
Effects of trigeminal nerve stimulation (TNS) on cerebral perfusion. A, Cerebral blood flow (CBF). B, Brain oxygen tension (Pbro 2). There was a significant decrease in CBF and Pbro 2 at the hemorrhagic shock (HS) onset (0 min). With TNS treatment, CBF and Pbro 2 significantly increased at 30 min after HS in comparison with the vehicle group. Data were expressed as mean ± sd and analyzed using one-way and two-way repeated measures analysis of variance. # p < 0.001 versus HS onset (0 min) in TNS group, % p < 0.001 versus HS onset (0 min) in the vehicle group, * p < 0.05 versus vehicle at the same time points, n = 6/group.

Effect of TNS on Catecholamine Release Following Severe HS

Norepinephrine levels were analyzed before, during, and after severe HS. In the vehicle group, blood norepinephrine levels were 8.4 ± 2.2, 9.8 ± 6.7, and 25.1 ± 17.9 ng/mL at the baseline, during HS (–15 min), and at HS onset (0 min), respectively (Fig. 5A). There was no significant difference when compared with the TNS group. With TNS treatment, norepinephrine levels were significantly increased by 339% when compared with the vehicle group (306.1 ± 87.2 vs 69.7 ± 35.9 ng/mL) at 15 minutes after the HS. These data demonstrate that TNS may elicit release of norepinephrine, thus protecting against the reduction of MAP and cerebral perfusion pressure induced by hypovolemia.

Figure 5.
Figure 5.:
Effects of trigeminal nerve stimulation (TNS) on catecholamine release and systemic inflammation following severe hemorrhagic shock (HS). A, Blood norepinephrine levels were measured before (baseline, –35 min), during (–15 min), onset (0 min), and after (15 min) severe HS. With TNS treatment, norepinephrine levels were significantly increased by 339.5% when compared with the vehicle group at 15 min after the HS. B, Blood levels of tumor necrosis factor (TNF)–α and interleukin (IL)–6 decreased significantly in the TNS treatment group compared with the control group at 15 and 30 min after severe HS. Data were expressed as mean ± sd and compared by one-way and two-way repeated analysis of variance and Student-Newman-Keuls test. # p < 0.05 versus baseline in TNS group, % p < 0.05 versus baseline in the vehicle group, * p < 0.05 versus vehicle at the same time points, n = 6~8/group.

Effect of TNS on Systemic Inflammation Following Severe HS

Severe HS triggers major systemic inflammatory response as associated by the measurement of blood proinflammatory cytokine levels. In the vehicle group, blood tumor necrosis factor (TNF)–α and interleukin (IL)–6 levels were increased 63.9x and 48.2x at 15 minutes and 64.7x and 131.8x at 30 minutes after HS compared with the baseline, respectively (Fig. 5B). With TNS treatment, their levels were significantly decreased: 40.3% and 22.6% for TNF-α and 69.2% and 80.7% for IL-6, at 15 and 30 minutes after HS when compared with the vehicle, respectively.

DISCUSSION

The optimal field resuscitative strategy after HS is controversial (45). Additionally, in combat and civilian situations where care is delayed or fluid resuscitation is logistically difficult, methods to prolong life have so far remained elusive. Therefore, novel resuscitation strategies that can avoid the complications of IV fluids and yet maintain adequate hemodynamics are highly desirable. In this proof-of-concept study, we have shown in an animal model of severe HS that an improved outcome from HS can be achieved by exploiting existing neural pathways and compensatory mechanisms with the electrical stimulation of the trigeminal nerve. To our knowledge, this is the first study where TNS has been explored as a resuscitative agent in HS.

The trigeminal nerve fibers are divided into three main branches with projections to both sympathetic and parasympathetic centers (21). Our results show that intermittent TNS elicits strong synergistic coactivation of the SNS and parasympathetic nervous system as analyzed by HRV. We demonstrate that LF/total increased 2.7x during TNS and decreased by 60% while maintaining higher MAP immediately after TNS. Concurrently, HF/total decreased by 50% during TNS and increased 1.3x after TNS. We further demonstrate that intermittent TNS readjusts the autonomic balance through attenuation of sympathetic hyperactivity paralleled by increase in parasympathetic tone, while maintaining augmented hemodynamics achieved during active stimulation following severe HS.

The neural circuit that is responsible for TNS-induced vasopressor response has been previously described (23). The sympathetic part of the efferent arm of the baroreflex originates in the rostral ventrolateral medulla (RVLM). The trigeminal nerve fibers project to RVLM, which plays a key role in determining peripheral sympathetic vasomotor tone and BP tone (24–27). Previous studies have shown that stimulation of trigeminal nerve with cold water, electricity, or ammonia vapor can activate this neural pathway (2829). Likewise, our results demonstrate intermittent, electrical TNS-induced, vasopressor response which results in improved hemodynamics and perfusion of ischemic prone organs after acute blood loss.

We further demonstrate that sympathetic nerve activity driven LF oscillations of BP was generated during TNS. The patterns of LF sympathetic oscillations have been shown to be highly associated with an increased tolerance to central hypovolemia (32030). The pronounced oscillatory patterns of circulatory pressure represent a sensitive sympathetically mediated “on-off” feedback mechanism designed to maintain tissue oxygenation despite the compromise in blood flow under severe hypovolemic conditions. We show that TNS triggers a tightly coupled and rhythmic relationship between BP and sympathetic outflow. Such increase in coherence reflects greater baroreflex modulation of sympathetic activity compared with the baseline state and thus may be beneficial in mounting an appropriate compensatory response to hypovolemia. The importance of TNS-modulated oscillatory patterns is highlighted by the fact that such kinds of tight coupling may be lost at the point of hemodynamic decompensation (31); it seems likely that TNS initiates such synchronization in order to delay the progression of irreversible shock. Our findings suggest that the way we stimulate the trigeminal nerve may mimic the neural traffic seen during the endogenously occurring slow oscillations which synchronize the discharge patterns of sympathetic neurons to increase tolerance to central hypovolemia, the detailed mechanism of which will be the subject of future investigation.

Cerebral ischemia that occurs when hemorrhagic hypotension is prolonged is a primary cause for mortality and morbidity following hemorrhagic injuries (32). Based upon previous studies, preservation of CBF and cerebral tissue oxygenation following HS are associated with increased tolerance to central hypovolemia and delayed onset of presyncope (33). Consistent with these studies, we observed a significant decrease in CBF and Pbro2 following severe HS; with TNS treatment, we showed significant increases in CBF and Pbro2. As discussed earlier, TNS causes peripheral vasoconstriction, but multiple studies have now shown that in the cerebral vascular bed, it has the opposite effect (34). There are four different pathways by which TNS can potentially improve CBF, the details of which have been previously described (1535–40). Our experimental results suggest that TNS causes disproportionate redirection of blood from the periphery to the brain even when there is severe loss of total blood volume. Such improved cerebral perfusion and oxygenation by TNS is another important mechanism that may contribute to increasing the tolerance to hypovolemic stress following severe HS.

Besides systemic hypoperfusion and ischemia, HS also induces inflammatory cascades, one of the key drivers of end-organ injury. Previous studies have shown that hypotension after HS, leading to ischemia and hypoxia, can provoke inflammatory cascades globally (4142). In this study, we show that blood levels of TNF-α and IL-6 were dramatically increased in both vehicle and TNS treatment groups after severe HS. However, TNS significantly decreased the TNF-α and IL-6 levels, which indicates that TNS may have antiinflammatory effects as well. The exact etiology for reduced inflammation in TNS group is unclear, although antiinflammatory effects of TNS have been reported before (1543). The reduced inflammation in TNS treatment group could be due to improved hemodynamics which results in attenuation of global hypotension and ischemia. A second possible explanation is that it is due to the activation of vagus parasympathetic fibers via the trigemino-vagal connection. The parasympathetic part of TNS is mediated by the dorsal motor nucleus (DMN) and nucleus ambiguous in the brainstem medulla oblongata. Trigeminal nerve fibers project to the DMN (44) which is a major parasympathetic outflow nuclei, and the nucleus of tractus solitarius which has wide connections to the preganglionic parasympathetic fibers (45). Animal studies of vagus nerve stimulation in HS have shown beneficial effects, which have been primarily attributed to vagus nerve’s antiinflammatory effects by blunting the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (45, 46). Consequently, it is reasonable to postulate that the beneficial effects of TNS in our study are due to integrated sympathetic and parasympathetic responses, with the SNS involved in maintaining BP and organ perfusion and the parasympathetic nervous system (PNS) involved in keeping shock-induced inflammatory cascades in check.

Some limitations of our study should be noted. First, although the pressure-controlled HS model described here represents a reproducible and realistic model, we observed that there was an individual variability in the compensatory response to blood loss and time course for the development of shock. To date, HS severity cannot be accurately assessed by one specific variable or index. Therefore, we selected the rats for analysis through a combined assessment of hemodynamic and metabolic (lactate; glucose) variables to make sure that the rats experienced a similar shock condition. Second, the way we inferred SNS and PNS activity was via HRV trends. Although the HRV gives important information regarding sympathetic and parasympathetic activities in a noninvasive way, the best way to determine these activities is to directly place electrodes on vagus and a sympathetic nerve (e.g., splanchnic, renal, etc) and record activity while the animal is undergoing TNS. Such kind of data could be of value in elucidating the mechanistic basis of the TNS effect on ANS activities that we have described in this report. The third limitation of this study is that the experiments were conducted in isoflurane anesthetized rats. Isoflurane has previously been reported to suppress parasympathetic activity (47). Therefore, the observed changes in HF/total and antiinflammatory effect are more likely to be underestimated in this study, especially when compared with unanesthetized animals. However, we do not expect isoflurane usage to significantly affect our result as animals in all groups were subjected to identical isoflurane exposure.

CONCLUSIONS

To our knowledge, this is the first time that TNS has been explored as a novel resuscitation strategy in an animal model of HS by exploiting endogenous compensatory autonomic mechanisms. The results of this study showed that the stimulation of trigeminal nerve interplays the SNS and parasympathetic nervous system activities to improve the survival.

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      Keywords:

      autonomic nervous system; hemorrhagic shock; neuromodulation; resuscitation; sympathetic nervous system; trigeminal nerve stimulation

      Supplemental Digital Content

      Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine and Wolters Kluwer Health, Inc.